Anxa2

Cells were lysed in 250 mM NaCl, 5 mM EDTA, 50 mM tris (pH 7.4), and 0.5% NP-40 containing protease inhibitors. After lysis, the lysate was spun at 15,000 rpm for 5 min. Samples boiled in SDS sample buffer containing reducing agents (Bio-Rad) were loaded and electrophoresed on a 4 to 12% bis-tris gel (Bio-Rad) for 2 hours at 120 V. The gels were transferred onto nitrocellulose membranes at 80 V for 1 hour at 4°C. The membranes were blocked in 5% bovine serum albumin (BSA) overnight at 4°C on a shaker. Primary antibodies were added in 2.5% BSA, and the membranes were incubated at room temperature for 2 hours. The membranes were washed and then incubated with rabbit or mouse secondary antibodies against horseradish peroxidase (1:5000; GE) for 1 hour at room temperature. The membranes were again washed and then developed using enhanced chemiluminescence reagent (GE).
Western blot analysis was performed using the following primary antibodies: a rabbit polyclonal antibody against Sema3D (1:1000; Abcam), a rabbit polyclonal antibody against AnxA2 (1:000; Santa Cruz Biotechnology), a rabbit polyclonal antibody against PlxnD1 (1:1000; Novus), or a mouse polyclonal antibody against β-actin (1:500; Santa Cruz Biotechnology).

AnxA2, Fam13A, Atg5, Atg7, Beclin1 and siNC siRNAs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). MH-S cells were transfected with siRNA (5 pM), LC3-RFP G120A, pcDNA3.1 and pcDNA3.1-Fam13A (100 ng) plasmids using LipofectAmine 2000 (Life Technologies) for 24 h following the manufacture’s instruction. In indicated case, AnxA2^−/− AM cells were treated with 5 μM autophagy activator Trehalose (Tre, Sigma-Aldrich), while WT AM cells were treated with 5 μM 3-Methyladenine autophagy inhibitor (3-MA, Sigma-Aldrich) for 2 h before and during PAO1 infection as indicated.

HEK293 cells (CRL-1573, ATCC) were transfected with pIRESneo-p11-Flag-HA (p11-WT) and pIRESneo-p11C83Q-Flag-HA (p11 C83Q mutant). Immunoprecipitation was performed with anti-Flag affinity gel (A2220, Sigma, St Louis, MO, USA) as described previously^{6 (link)}. Immunoblotting was performed with a standard protocol using the following antibodies: mGluR5 (rabbit monoclonal (1:5000), Abcam, Cambridge, MA, USA), AnxA2 (mouse monoclonal (1:1000), sc-28385, Santa Cruz), p11 (for human p11, mouse monoclonal (1:1000), BD Bioscience, San Jose, CA, USA; for mouse p11, goat polyclonal (1:200), R&D systems, Minneapolis, MN, USA), PSD95 (mouse monoclonal (1:2500), clone K28/43, Millipore, Billerica, MA, USA), mGluR2/3 (rabbit polyclonal (1:5000), Millipore) and GAPDH (mouse monoclonal (1:2500), clone 6C5, Chemicon, Billerica, MA, USA). Immunoblot analysis was performed with Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA).